|Patrik Appelblad1; Albena, Mihailova2; Hans-Ake Lakso3; Jorn Schneede3|
1Merck SeQuant AB, Umeå, Sweden; 2Department of Clinical Chemistry, Akershus
university Hospital, Lorenskog, Norway; 3GKM/Laboratory medicine, University
hospital, Umeå, Sweden
|A clinically useful indicator of cobalamin status in cobalamin-deficient individuals is the increase of methylmalonic acid (MMA) in serum or plasma. Total homocysteine, another biomarker for cobalamine-deficiency, is a less specific indicator of vitamin B12 function, because diverse genetic and lifestyle factors and disease states are influencing its concentration. In previous studies, MMA quantitation has been accomplished by GC-MS, capillary electrophoresis with fluorescence detection, and LC–MS/MS in serum or plasma samples. All previous methods have drawbacks, either being laborious yielding low sample throughput, being time consuming and expensive wherefore alternatives are still requested. Herein we present a simple and fast, yet rugged and reliable method for quantitation of methylmalonic acid in human plasma using isotopically labelled internal standards (d3-MMA), and negative ESI-MS detection mode after isocratic hydrophilic interaction liquid chromatographic (HILIC) separation on a zwitterionic silica stationary phase. |
Optimal chromatographic separation conditions were reached whilst using 80 volume- % acetonitrile and 20 volume-% aqueous ammonium acetate 100 mM (pH 4.5 and 20 mM total ionic strength). Experimental design followed by multivariate analysis was applied in order to find the best experimental conditions for the sample work-up. A simple sample preparation procedure based on protein precipitation followed by centrifugation prior to analysis was developed, and using this procedure, total time spent for sample work-up is roughly ten minutes per batch of samples. As the total chromatography cycle-time is ten minutes, the total work-flow may be considered as continuous. Analytical data have been produced to determine repeatability, reproducibility, precision, linearity, LOD and LOQ for MMA, using standard solutions, spiked plasma samples, controls, and human patient samples. The variation within-day, between-day as well as long term stability have all been monitored. The new method was compared to existing GC-MS and RPLC-MS/MS methodology using an adequate number of patient samples. Excellent sensitivity and adequate linearity (r2 > 0,999) are achieved with the LC-MS system set-up, and where LOQ for MMA is approximately 90 nM and LOD less than 30 nM for plasma samples. Transferring and re-validating the method on a LC-MS/MS platform was straightforward, and the corresponding figure of merits was comparable. As the endogenous levels for MMA are ranging between 100-500 nM, necessary target levels have been reached that allow this new method to be applied within clinical monitoring.